Tetracycline complexes and the use thereof in preparing and isolating tetracyclines



United States Patent 3,280,188 TETRACYCLINE COMPLEXES AND THE USETHEREOF IN PREPARING AND ISOLATING TETRACYCLINES Joao Villax, Travessado Ferreiro 3, Lisbon, Portugal No Drawing. Filed Mar. 19, 1964, Ser.No. 353,235 8 Claims. (Cl. 260559) The present invention relates to thepreparation and isolation of various tetracyclines such as tetracycline,oxytetracycline, chlortetracycline and dimethylchlortetracycline, bymeans of new alkaline earth metal dibenzylideneethylenediimine complexesof the respective tetracyclines.

In the embodiment of the present invention the new complexes are formedby adding N,N-dibenzylideneethylenediimine, hereinafter designated byA-DBE'D of the formula to the culture medium during fermentation or tothe already fermented broth, in the presence of alkaline earth metalions, and are precipitated after separation from the myceli-um at a pHbetween 8 and 9.5. The respective tetracycline is then liberated fromthe complex thus obtained, by adjusting the pH to its isoelectric pointby acidification.

The present invention presents important industrial advantages:isolation accomplished in few steps; organic solvents not used; use ofcheap isolation products; and high yield.

When A-DBED is added to the culture medium during fermentation, thefermentation yield, i.e. the antibiotic activity attained by the mediumduring fermentation, increases by to 20% approximately, according to theconditions and the strain used, in comparison to parallel fermentationsrun without addition of A-DBED.

The ouantity of A-DBED added to the broth is. at least, half a mole permole of antibiotic, in respect of tetracycline, oxytetracycline andchlortetracycline. In the case of demethylchlortetracycline, it ispreferable to use one mole and a half of A-DBE-D per mole of theantibiotic.

In order to obtain the highest yields, there are added duringfermentation to 1000 milligrams of A-DBED per liter of broth in variousportions, and to the acidified and filtered broth there is added, at pH5, the quantity of A-DBED necessary to obtain the above-mentionedproportions, calculated on the quantity of antibiotic formed duringfermentation. The embodiment of the present process also includes theisolation of the tetracyclines by means of the A-DB-ED complexes, in theevent of the same being carried out exclusively in the phase of thealready fermented broth.

Any tetracycline-producing microorganism, such as Streptomycesalboflavus, Sterptomyces aureofaciens, Streptomyces flavus,Streptomyces, flaveolus, Streptomyces rimosus, Streptomycesviridifaciens, Slreptc-myces sayamaensis, Streptomyces psam m'oticus,Streptomyces lusiranus, Streptomyces varsoviensis, including itsmutants, can be used in the present process.

The A-DBED is added to the culture medium in various portions understerile conditions. When the additions are made at 0, 3'6, 96 and 120hours of fermentation, satisfactory results are obtained. The fermentedbroth is then acidified at a pH between 0.5 and 1.8 with an acid, suchas sulphuric and oxalic acid, although other acids can also be used. Thebroth is then filtered and the pH adjusted to 5 With ammonia, in theevent it contains alkaline earth metal ions in quantity sufficient toform the A-DBED complex, and if not the pH is adjusted with an alkalineearth metal hydroxide. There is then added the quantity or" A-DBEDnecessary to obtain a total quantity of, at least, half a mole per moleof the tetra cycline, oxytetracycline or chlorotet-racycline present,and at least one mole and a half per mole of demethylchlortetracycline.After stirring the addition of the base is continued until pH 8 to 9.5.The A-DBED complex is then precipitated.

Analysis confirms that the complexes formed correspond to the followingformulas:

Tetracycline .A-DBED .Me, Oxytetracycline .A-DBED .Me .161-1 0Chlortetracycline,,A-DBED .Me Demethylchlortetracycline .A-DB'ED .Me

where Me is an alkaline earth metal. In addition to the above-mentionedcomplexes, other less important complexes having a different compositioncan he prepared and formed.

The complex formed is then filtered oif, Washed with water andsuspended, when still wet, in water. By adjusting the pH of thesuspension to the isoelectric point of the respective tetracycline withan acid such as hydrochloric, sulphuric or oxalic acid, thecorresponding tetracycline base can be prepared in pure state. Byrecrystallizing the complexes, using a suitable solvent, such asdimethylformamide, by addition of water, the complexes can be obtainedin pure form.

In view of the fact that the 'br-oths used usually contain calcium saltsin a great amount and the broth acidified by dilute sulphuric acid alsocontains calcium ions in excess for the precipitation of the complex,the addition of a sequestering agent, such as ethylenediaminetetraacetate, before the precipitation of the complex, makes it possibleto obtain higher yields. Also the addition of an antioxidant to theacidified broth makes it possible to obtain a product of a higher degreeof purity. As antioxidant, ammonium formaldehyde sulfoxylate ispreferred but any other smooth antioxidant can be used.

In each case, the base obtained after precipitation at the pH of theisoelectric point meets the degree of purity required by the variouspharmacopeias and can be used for medicinal purposes or can betransformed into the corresponding acid salts by known methods.

The other fermentation conditions are generally known and the followingexamples give a full illustration of how same is performed.

The A-DBED complexes, although used essentially for isolating therespective tetracyclines, can also be used as such for pharmaceuticalpurposes, having an antibiotic activity in vitro and in vivo equivalentto the respective tetracycline they contain. They can also be used asfeed supplement for animals.

The various A-DBED complexes present in pure state the followingcharacteristics:

A-DBED .Ca.,.tetracycline decomposition over 200 C.; specific rotation:[a] =225 (c.=l in N/lO HCI). Ultraviolet spectrum:

A max. 222 (Elfi 292); x max. 255/56 (Ei'fi' max. 355/357 (EV 261) Amax. 254 (Eli g 400); A max. 353 (Elfi 231) Infrared spectrum having thefollowing maxima in Nujol: 301 6.12 1. (shoulder), 6.22 (shoulder), 6.321, 631

3 6.93a, 7.17 7.66 7.9;, 807 822a, 859 8.94a, 9.21 9.35 1,, 9.52 9.729.92;, 10.35 1067 11.66;, 1195a, 1235p.

A-DBED .Ca chlortetracycline decomposition beginning at 200 C.; specificrotation: =230 (c.=1 in N/ 10 HCl). Ultraviolet spectrum:

A max. 232. (EPZ 375); A max. 255 (E12,, 376); A max.

370 (Efi 178) Infrared spectrum having the following maxima in Nujol:295 612 (shoulder), 6.26a, 6.35 (shoulder), 6.80, (shoulder), 6.9 7.15(shoulder), 7.62 1, 8.17 (shoulder), 8.30 1, 8.60;, 8.92 1, 9.57,u,10.28,.4, 1215a, 12.37 1.

A-DBED .Ca .demethylchlortetracycline 1 decomposition over 200 C.;specific rotation: [a] =180 (c.=1 in N/ HCl). Ultraviolet spectrum:

A max. 232 (Efg 503); X max. 366/368 (Ei'fi 171) All media were preparedwith tap water. A fermenter having a useful capacity of 6000 liters,containing a medium of the following composition:

Grams per liter of tap water Corn steep liquor 28 Calcium carbonate 14Starch 38 (NH SO 5.7 NH C1 1.5 MDSO4.4H2O 0.05 C'OClg.6H2O ZnS=O 0.05Peanut meal 25 Lard oil 35 (pH 6.76.8 after sterilization.)

is inoculated with 150 liters of a pre-culture of Streptomycesviridifaciens. There is added to the medium 1 gram/ liter of A-D BEDdivided into four equal portions at 0, 36, 72. and 98 hours offermentation. The fermentation is performed at 28 C. with aeration of 2liters/ liter per minute. After 150 hours, 8.6 grams per liter oftetracycline are obtained. The medium is then acidified to pH 1.5 with25% sulphuric acid, filtered with a drum filter and the cake is thenextracted twice with water at pH 1.5. There is then added to the unitedfiltrate 18 kilograms of ethylenediamine tetraaceta-te as sequesteringagent and 12 kilograms of A-D-BED; and then the pH is adjusted to 9.5with 12% ammonia. After 3 hours of agitation, the precipitate whichconsists essentially of impure AD-BED-tetracycline calcium complexhaving the formula A-DBED .Ca .tetracycline is filtered 01f. The wetprecipitate is then suspended in water, acidified with .a 10% aqueoussolution of oxalic acid to pH 1.5 under agitation. Then the solution isfiltered and the pH is adjusted to 5.8 with a 10% aqueous sodiumhydroxide solution. The tetracycline base precipitates. The precipitateis filtered and dried under reduced pressure at 65 C. Effective yield88% calculated in activity.

4 Example 2 1 liter of sterilized medium having the composition:

Corn steep liquor 50% grams 10 Sugar do 10 CaCO do 1 (NHgJzI'EPO; do 2KH 'PO do 2 MgSO .7 H O do 0.25 Water milliliters 1000 was inoculatedwith 1 milliliter of spore suspension of Streptomyces alboflavus mutantM108-OX (CBS) and incubated at 25 C. in a rotary shaker for 36 hours.Afterwards, a fermenter containing a sterilized medium of the formula:

was inoculated with the above-mentioned pre-culture of 36 hours. 400milligrams per liter of A-DBED divided into four fractions are added at0, 36, 72 and 96 hours of fermentation.

Submerged fermentation was then performed at 26 C. under stirring andsterile aeration. Fermentation was stopped after 150 hours, showing afinal concentration of oxy-tetracycline of 7. 6 grams per liter.

The fermented broth is acidified with sulphuric acid (25 until pH 1.5,filtered and the mycelium washed with water. The joint filtrate has avolume of 240 liters. There is then added 2 grams per liter ofethylenediamine tetraacetate (sequestering agent). The pH is thenadjusted to '6 with ammonium hydroxide (12% grams of A-DBED added andthe pH raised to 9.5 with ammonium hydroxide (12%). After stirring for 3hours, the formed precipitate is filtered and washed until the pH of thewashing waters reaches pH 7. The wet precipitate is suspended in waterand acidified with oxalic acid (10%) to pH 1.5. The solution is thenfiltered and the pH adjusted to 5. The pure oxytetracycline basecrystallizes. After filtering and washing, the product is dried.Effective yield 87%.

Example 3 The procedure is as in Example 2, but inoculation of themedium is with Streptomyces rimosus. 142 hours later, the broth contains6.3 grams per liter. Final yield 89%.

Example 4 300 liters of a sterile culture medium of the (pH 6.7 afitersterilization.)

are inoculated with a spore suspension of Streptomyces lusiranus 101-A(CBS) and 1200 milligrams per liter of A-DBED divided into fourfractions are added to the medium at 0, 36, 72 and 96 hours. Thefermentation is performed at 27 C., under stirring and sterile aeration,gradually increasing from 1.5 liters per liter per minute to 4.0 litersper liter per minute. After 150 hours the activity in chlortetracyclineis 12.4 grams. The broth is then acidified with sulphuric acid (25%) topH 0.5 and filtered. Then the pH is raised to with ammonium hydroxide(12%) and 600 grams of A-DBED, 1.5 grams per liter of ethylenediaminetetraacetate and 0.3 gram per liter of sodium sulfite added to thefiltrate. Upon raising the pH to 8.7, the A-DBED complex precipitates.After stirring for 2 hours, the precipitate is filtered, washed withWater and well drained. The still Wet precipitate is added to 12 litersof dimethylformamide and the complex precipitated by adding 36 liters ofwater, adjusting the pH to 8.5. The precipitate is filtered off, washedwith water until the washing Water reaches a neutral pH, and the productdried. There is obtained the A-DBED-chlortetracycline complex,containing according to analysis, 19.5% of chlortetracycline expressedas anhydrous base, 4.89% calcium, 14.8% A-DBED and 1.1% Water. The invitro antibiotic activity is 863 micrograms per milligram expressed aschlortetracycline hydrochloride. Effective yield 8 8.1%.

Example 5 300 liters of medium of the formula specified in Example 4 areinoculated with a spore suspension of ademethylchlortetracycline-producing strain of Streptomyces aureofaczens.There are added to the medium 100 milligrams per liter at the beginningand 200-200 milligrams per liter of A-D BED after 56 and 112 hours offermentation. After 130 hours of fermentation, 2.8 grams per liter ofdemethylchlortetracycline are obtained. To the acidified filtrate 170grams of A-DBED are added, and the further procedure is as in Example 4.The final product has a composition of: 52.8% ofdemethylchlortetracycline as anhydrous base, 38.4% of A-DBED, 6.72% ofcalcium and 2.43% of water and corresponds to the formula:Demethylchlortetracycline .A-DBED .Ca in anhydrous state. Yield 79%.

Having thus disclosed the invention What is claimed is:

1. The -N,-N-dibenzylideneethylenediimine complex of tetracycline of thecomposition:

Tetracycline A-DBED .Me

where Me is an alkaline earth metal ion.

2. The N,N-dibenzylideneethylenediimine complex of oxytetracycline ofthe composition:

Oxytetracycline .A-DBED .Me .16H O Where "Me is an alkaline earth metalion.

3. The -N,N-dibenzylideneethylenediirnine complex of chlortetracyclineof the composition:

Chlortetracycline .A-D BED .Me

6. The N,N-dibenzylideneethylenediimine complex of oxytet-racycline ofthe composition:

Oxytetracycline .A-DBED .Ca .16H O 7. The.N,N'-dibenzylideneethylenediimine complex of chlortetracycline of thecomposition:

chlortetracycline .A-DBED .Ca

8. The N,N'-dibenzylideneethylenediimine complex ofdemethylchlortetracycline of the composition:

Demethylchlortetracycline .A-DBED .Ca

References Cited by the Examiner UNITED STATES PATENTS 2,734,0 1'82/1956 Minieri et al. 80 2,739,924 3/ 1956 Lein et al. 195-80 2,791,6095/1957 Kaplan 260559 2,915,555 12/1959 Solomons 260559 ALEX MAZEL,Primary Examiner. HENRY R. JILES, Examiner.

JAMES W. ADAMS, 111., Assistant Examiner.

1. THE N,N''-DIBENZYLIDENEETHYLENEDIIMINE COMPLEX OF TETRACYCLINE OF THECOMPOSITION: